Biosynthesis of Co3O4 nanomedicine by using Mollugo oppositifolia L. aqueous leaf extract and its antimicrobial, mosquito larvicidal activities

Nanotechnology is a relatively revolutionary area that generates day-to-day advancement. It makes a significant impact on our daily life. For example, in parasitology, catalysis and cosmetics, nanoparticles possess distinctive possessions that make it possible for them in a broad range of areas. We utilized Mollugo oppositifolia L. aqueous leaf extract assisted chemical reduction method to synthesize Co3O4 nanoparticles. Biosynthesized Co3O4 Nps were confirmed via UV–Vis spectroscopy, scanning electron microscope, X-ray diffraction, EDX, Fourier-transform infrared, and HR-TEM analysis. The crystallite size from XRD studies revealed around 22.7 nm. The biosynthesized Co3O4 nanoparticle was further assessed for mosquito larvicidal activity against south-urban mosquito larvae Culex quinquefasciatus, and antimicrobial activities. The synthesized Co3O4 particle (2) displayed significant larvicidal activity towards mosquito larvae Culex quinquefasciatus with the LD50 value of 34.96 µg/mL than aqueous plant extract (1) and control Permethrin with the LD50 value of 82.41 and 72.44 µg/mL. When compared to the standard antibacterial treatment, Ciprofloxacin, the Co3O4 nanoparticle (2) produced demonstrates significantly enhanced antibacterial action against the pathogens E. coli and B. cereus. The MIC for Co3O4 nanoparticles 2 against C. albicans was under 1 μg/mL, which was much lower than the MIC for the control drug, clotrimale, which was 2 µg per milliliter. Co3O4 nanoparticles 2, with a MIC of 2 μg/mL, has much higher antifungal activity than clotrimale, whose MIC is 4 μg/mL, against M. audouinii.

www.nature.com/scientificreports/ acid, causing cell death by interfering with normal functioning like replication. The mechanism of action of AgNPs towards larvae is poorly understood, with just a small number of publications available [5][6][7][8][9] . The effects of NPs on mosquito larvae were studied by Kumar et al. in terms of morphological, biochemical, physiological, and molecular alterations 10 . Infectious illnesses, in general, constitute a severe danger to public health across the globe, particularly when antibiotic-resistant probiotic pathogens evolve. Gram-positive and Gram-negative strains of bacteria are both regarded to be a substantial public health concern. Antibiotics were used to manage diseases in both the community and hospital settings for many years [11][12][13] .
Mollugo oppositifolia L., often known as slim carpet weed, belongs to the Molluginaceae species (English). It's a thin, widespread, sleek, branching annual plant with 20 to 30 cm high branches that thrives in both dry and wet environments. Leaves are alternate or leathery, in tendrils of 4-5, uneven, oblanceolate or linear-lanceolate or occasionally rounded or sharp and apiculate at the apex, greatly tapering into the inconspicuous petiole. Flowers are white and borne in two or more axillary fascicles. Capsules are ellipsoid in shape and contain many dark brown seeds. Creaper and undesired roots 14 . In ethnomedicine, the herb is used for stomachic, earache, aperients, and skin problems. The leaves have a harsh flavor and are antiseptic. Mollugo subspecies have been shown to have antibacterial, anticancer, anti-inflammatory, and hepatoprotective properties 15 .
Due to their fascinating properties, nano-structure materials have drawn significant attention in recent years. Among these elements, much focus is drawn to research on fundamental characteristics and functional applications of transition-metal oxides [16][17][18][19] . Among the transition-metal oxides, cobalt oxides, Co 3 O 4 and CoO are flexible materials that are stable in the natural environment 20,21 . In recent years, owing to their possible applications, much effort has been guided towards the synthesis and investigation of Co 3 O 4 and CoO nanostructures [22][23][24] . Co 3 O 4 is the thermodynamically stable type of cobalt oxide below 1164 K in ambient air, while Co 3 O 4 is decomposed into CoO above this temperature 25 . Co 3 O 4 is a natural spinel 26 at room temperature and has several possible applications in gas sensors, magnetic materials, catalysts, and absorbers of solar energy [27][28][29][30] . Several processes, such as oxidation, microwave-assisted hydrothermal, ultrasonic, and hydrothermal have recently been developed for the preparation of Co 3 O 4 [31][32][33][34] . CoO, on the other hand, crystallises in the structure of rock salt and has possible uses in many areas, such as lithium battery anodes, pigments, magnetoresistant reading heads, and gas sensors [35][36][37] . While there are a few studies on the synthesis of CoO in bulk form, through simple methods, this compound is hard to acquire in pure form, mostly polluted with Co 3 O 4 and Co metal.
Cobalt nanoparticles (Co NPs) have gained a lot of interest recently owing to their unique electrical and magnetic characteristics and lower cost compared to noble metal nanoparticles (NPs) 38,39 . Biomedical researchers have investigated the potential of CoNPs as therapeutic agents for the therapy of disorders like microbial infection 40,41 . At low concentrations, CoNPs are safe for the body, have potent antimicrobial and antifungal activities, and fewer adverse effects than antibiotics 42,43 .
Yin and Wang demonstrated that in the existence of surfactant Na(AOT) at 130 °C in air, decomposition of Co 2 (CO) 8 47 . A basic synthesis strategy, different from those described above, is suggested in this article. Through green chemistry approach Mollugo oppositifolia L. aqueous leaf extract assisted strategy, we will report on the synthesis process of Co 3 O 4 nanoparticles and evaluation of its mosquito larvicidal and antimicrobial activities.

Results and discussion
Physiochemical characterization of biosynthesized Co 3 O 4 NPs. The detailed schematic representation of biosynthesized Co 3 O 4 nanoparticles is shown in Fig. 1a. FTIR spectra are often collected between 400 and 4000 cm −1 . The FT-IR spectra of the biosynthesized Co 3 O 4 NPs are shown in Fig. 1b. A wide peak at 3465.93 cm −1 indicates the presence of the N-H group, which may have appeared as an amine moiety. The presence of C-H functional group alkanes is indicated by a band between 2800 and 3000 cm −1 . C=O was identified at 1644.01 cm −1 from the PVP moiety, as shown by spectral peaks. Both tetrahedral and octahedral Co-O vibrations are confirmed by the bands at 509.59 cm −1 and 584.80 cm −1 , respectively. The functional groups of the capping agent and the synthesis of Co 3 O 4 nanoparticles were authenticated by FT-IR analysis. In addition, The XRD pattern was employed to analyze the phase purity and crystalline nature of biosynthesized Co 3 O 4 NPs, as shown in Fig. 1c.
Pure face-centered cubic spinel phase structure of Co 3 O 4 NPs was identified by diffraction peaks at 2 = 31.2°, 37.6°, 38.7°, 44.8°, 55.6°, 59.8°, and 66.3°, which were indexed to (220), (311), 222), 400, (422), 511, and 440) planes. Standard Co 3 O 4 NPs were found to have diffraction peaks that were quite similar to those produced. The diffraction peaks all rather closely match the typical distribution for pure Co 3 O 4 nanoparticles (JCPDS No. 00-042-1467). Certain peaks indicative of impurities have been detected. These pronounced peaks show that the resulting nanoparticles are very crystalline. The average crystallographic size may be determined from the observed primary diffracted peak by using the Scherer equation, where, D (hkl) is the typical crystallographic dimension, k is shape constant (0.89), λ is the wavelength of the incident x-ray (Cukα source, λ = 0.15405 nm), β is the full width half maximum (FWHM), θ is the incident angle of x-ray. A 22.70 nm Co 3 O 4 crystal was successfully produced.

Morphological and elemental analysis of biosynthesized Co 3 O 4 Nps.
Scanning electron microscopy was used to regulate the resulting Co 3 O 4 NPs' size and form (SEM). Scanning electron microscopy (SEM) scans confirmed the spherical shape of the biosynthesized Co 3 O 4 NPs ( Fig. 2a-c). The biosynthesized Co 3 O 4 NPs were distributed in the wild as a population of uniformly sized particles. In addition, the EDX analysis confirmed the atomic composition of the biosynthesized Co 3 O 4 NPs. The presence of cobalt and oxygen peaks in the EDX spectra confirmed that the material was really Co 3 O 4 NPs (Fig. 2d). There was 3.58% cobalt and 64.20% oxygen by molecular weight. Extra peaks in the EDX spectra might be due to bioorganic or contaminant presence in the solution. The chemical composition of the biosynthesized Co 3 O 4 nanoparticles is shown in Fig. 2e. Studies using scanning electron microscopy to map the nanoparticle proved its identity as Co 3 O 4 (Fig. 2f). Cobalt is represented by the pink dots, whereas Oxygen is shown by the green ones. Detailed morphological features and chemical compositions of biosynthesized Co 3 O 4 nanoparticles were analyzed by HR-TEM, obtained results are shown in Fig. 2g-j. TEM images demonstrate the existence of aggregated polycrystalline particles with restricted size distribution and a spherical shape. The particle size from TEM images is well-matched with the particle size predicted by the Debye-Scherrer equation. Figure 2j shows the elemental mapping of biosynthesized Co 3 O 4 nanoparticles, which confirms the presence of Co and O elements with uniform distribution.
Larvicidal activity. The biosynthesized Co 3 O 4 particle (2) was much more active in contrast to Culex quinquefasciatus with an LD 50 value of 34.96 μg/mL than the aqueous plant extract (1) and control Permethrin, which had LD 50 values of 82.41 and 72.44 μg/mL, respectively. The aqueous plant extract (1) showed the least amount of activity against Culex quinquefasciatus, with LD 50 values that were respectively 82.41 μg/mL. This was one of the samples that was tested. When compared to the positive control Permethrin, which had an LD 50 value of 72.44 μg/mL, the manufactured Co 3 O 4 nanoparticle (2) exhibited very high levels of activity, while the aqueous plant extract (1) exhibited only moderate levels of activity. The findings are shown in Table 1 below.
In vitro antibacterial activity. The antibacterial activity of ciprofloxacin was tested in vitro against four different bacteria: two Gram-negative (E. coli and Pseudomonas aeruginosa) and two Gram-positive (S. aureus  Antifungal activity. Fresh leaf extract 1 of Mollugo oppositifolia L. and synthetic nanoparticle 2 were both examined for their ability to inhibit the activity of four different types of fungi. When compared to compound 1, compound 2 is substantially more effective against fungi. Compound 2 effectively combats the fungal infections caused by Candida albicans and Malassezia audouinii. The minimal inhibitory concentration (MIC) of Compound 2 for C. albicans growth was 01 μg/mL, which was much lower than the control clotrimale (02 μg/ mL). Compound 2 has an even lower MIC against M. audouinii than clotrimale, which has a MIC of 4 μg/mL. You can see the results in Table 3 and Fig. 4.

Biosynthesis of cobalt oxide nanoparticles. Preparing cobalt oxide nanoparticles began with dissolv-
ing CoCl 2 ⋅6H 2 O (0.1 g) in a sufficient amount of deionized water, followed by the addition of 10 mL of a solution containing an extract of the Mollugo oppositifolia L. plant. Then, for 3 h at room temperature, the mixture was agitated at a speed of 1000 rpm using a magnetic stirrer. The pH of the reaction mixture was adjusted by adding a 1 mL solution of 10% NaOH to the mixture. The precipitate was filtered and then evaporated for 12 h, the oven was set to 150 °C and let to do its thing. Following collection, the powder was calcined for 3 h at 500 °C before being ground into a fine powder.   Antibacterial activity. Kirby Bauer tested the antibacterial effectiveness of an aqueous plant extract (1) and a mixed Co 3 O 4 nanoparticle suspension against Staphylococcus aureus, Escherichia coli, Klebsiella pneumophila, and Pseudomonas aeruginosa in vitro (2). Discs are the preferred method for dispersing molecules 48 . The antibacterial activity of ciprofloxacin was utilized as a standard. Bacteria were cultured on petri plates using nutrient agar. All of the synthesis was carried out in DMSO, and the chemicals were held on a filter paper disc that measured 5 mm in diameter and 1 mm in thickness. After 24 h incubation at 37 °C, the discs were tested for antibacterial activity by measuring the size of the inhibitory zone 49,50 surrounding each one put on the plates that had previously been implanted. Minimum inhibitory concentrations (MIC) were used to compare the antibacterial activity of an aqueous plant extract (1) and Co 3 O 4 nanoparticles (2).
Antifungal activity. The standardized disc-agar diffusion technique 51,52 was used to assess the antifungal activity of aqueous plant extract (1) and combined Co 3 O 4 nanoparticle (2). Microsporum audouinii (MTCC-8197), Candia albicans (MTCC-227), Cryptococcus neoformans (recultured) and Aspergillus niger (MTCC-872) were used to test antifungal activity. The materials were sterilised by filtering using 0.22 m Millipore filters after being dissolved in 10% dimethyl sulfoxide (DMSO) to a desired concentration of 30 mg/mL. Antifungal studies were then performed utilising disc diffusion technique with 100 L of solution containing 104 spore/mL of fungi dispersed over PDA medium. The discs (6 mm in diameter) were treated with 10 mL of the samples (300 g/ disc) and put on the infected agar. The typical medicine was clotrimale. 10 percent DMSO was used to make negative controls. For fungus specimens, the inoculation plates were then incubated at 37 °C for 72 h. Fungi linked with plants were cultured at 27 °C. The zone of inhibition against the tested strains was used to assess antifungal activity. In this study, each test was carried out twice.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.